Antibody based reagents that specifically recognize neurodegenerative disease related forms of the protein tdp-43

ABSTRACT

The invention relates to antibodies, antibody fragments and binding agents that specifically recognize TDP-43 associated with frontotemporal dementia (FTD), but not TDP-43 associated with amyotrophic lateral sclerosis (ALS) or TDP-43 associated with healthy human brain tissue, and antibodies, antibody fragments and binding agents that specifically recognize TDP-43 associated with ALS, but not TDP-43 associated FTD or TDP-43 associated with healthy human brain tissue.

RELATED APPLICATION

This application claims the benefit under 35 U.S.C. §119(e) of U.S.Provisional Application No. 61/934,443, filed Jan. 31, 2014, the entirecontents of which is hereby incorporated by reference.

FEDERALLY SPONSORED RESEARCH OR DEVELOPMENT

This invention was made with government support under AG042066 awardedby The National Institutes of Health. The government has certain rightsin the invention.

BACKGROUND OF THE INVENTION

Protein misfolding and aggregation is a common thread behind manyneurodegenerative diseases, including AD, Parkinson's disease (PD),Frontal Temporal Dementia (FTD), Lewy Body Dementia (LBD), andHuntington's disease (HD) among others. While each disease has beenprimarily associated with aggregation of a specific protein;beta-amyloid (Aβ) with AD, alpha-synuclein (a-syn) with PD and LBD, tauwith various tauopathies including AD and FTD, TDP-43 with amyotrophiclateral sclerosis (ALS) and FTD, and huntingtin with HD, more than oneprotein is likely to misfold and aggregate in brain tissue complicatingdiagnosis and treatment strategies. While all these proteins can formfibrillar aggregates, they can also form a variety of different smallersoluble aggregate structures as well, and increasing evidence implicatessmall soluble oligomeric forms of these different proteins as therelevant toxic species in the various diseases rather than the fibrillaraggregates that serve as diagnostic hallmarks. Since cellular stressinduced by misfolding and aggregation of one protein such as Aβ may welllead to misfolding and aggregation of other proteins such as tau anda-syn, the presence of multiple misfolded proteins in different diseasesshould be expected. Therefore characterizing which aggregated proteinspecies are present at different stages of each disease would greatlyfacilitate identification of suitable biomarkers and development ofbetter diagnostic and treatment strategies for these neurodegenerativediseases.

Accordingly, there exists the need for new therapies and reagents forthe diagnosis and treatment of frontotemporal dementia (FTD) andamyotrophic lateral sclerosis (ALS).

SUMMARY OF THE INVENTION

Variants of TDP-43 have been correlated with neurodegenerative diseasesincluding Alzheimer's, Frontotemporal dementia and ALS (Lou Gehrig'sdisease). Reagents that can selectively recognize disease relatedvariants have potential application as diagnostic and therapeutics forthese diseases. The reagents also have potential value as imaging agentsfor specific diseases.

Antibody fragments or nanobodies have been isolates that selectivelyrecognize variants of the protein TDP-43, a DNA binding protein that isinvolved in neurodegenerative disease. We have isolated nanobodies thatselectively recognize TDP-43 variants that occur in cases ofFrontotemporal Dementia and other nanobodies that selectively recognizeTDP-43 variants that occur in cases of amyotrophic lateral sclerosis(ALS or Lou Gehrig's disease). Variants of TDP-43 have also beenimplicated in Alzheimer's disease.

The present invention provides in certain embodiments an antibody orantibody fragment that specifically recognizes TDP-43 associated withfrontotemporal dementia (FTD), but not TDP-43 associated withamyotrophic lateral sclerosis (ALS) or TDP-43 associated with healthyhuman brain tissue.

The present invention provides in certain embodiments an antibody orantibody fragment that specifically recognizes TDP-43 associated withamyotrophic lateral sclerosis (ALS), but not TDP-43 associated withfrontotemporal dementia (FTD) or TDP-43 associated with healthy humanbrain tissue.

The present invention provides in certain embodiments an antibody orantibody fragment isolated according to a method comprising the stepsof:

-   -   (a) negative panning a scFV phage library comprising serially        contacting phage with:        -   (i) a generic protein and/or α-synuclein (α-syn); and        -   (ii) TDP-43 from healthy human brain tissue;    -   until less than 5% of the phage is observed binding to antigen,        which produces an aliquot of phage;    -   (b) positive panning of the aliquot from step (a) comprising        contacting the aliquot of phage from step (a) with TDP-43        associated with ALS or FTD, and incubating for time sufficient        to allow binding of phage to the brain derived TDP-43 associated        with ALS or FTD; and    -   (c) eluting the bound phage particles from step (b). In certain        embodiments, the step of observing of the binding of the phage        to the antigen is by using Atomic Force Microscope (AFM)        Imaging.

In certain embodiments, the antibody fragment is isolated according to amethod comprising the steps of:

-   -   (a) a negative panning of a scFV phage library wherein the        negative panning eliminates phage that bind to non-desired        antigens wherein the negative panning comprises serially        contacting phage with:        -   (i) a generic protein and/or α-synuclein (α-syn); and        -   (ii) TDP-43 from healthy human brain tissue;    -   and monitoring the binding of the phage to the generic protein        and the TDP-43 from healthy human brain tissue using Atomic        Force Microscope (AFM) Imaging and repeating steps (i) and (ii)        until no phage is observed binding to antigen by the AFM imaging        to produce an aliquot of phage;    -   (b) contacting the aliquot of phage with TDP-43 associated with        ALS or FTD and incubating for time sufficient to allow binding        of phage to the oligomers; and    -   (c) eluting the bound phage particles from step (b).

In certain embodiments, the TDP-43 associated with ALS or FTD is TDP-43associated with ALS. In certain embodiments, the TDP-43 associated withALS or FTD is TDP-43 associated with FTD.

In certain embodiments, generic protein is bovine serum albumin (BSA).

In certain embodiments, the negative panning is repeated until less than0-10% phage was observed by AFM imaging as binding to antigen in step(a).

In certain embodiments, the antibody fragment does not contain theconstant domain region of an antibody.

The present invention provides in certain embodiments an antibodyfragment comprising an amino acid sequence encoded by a nucleic acid,wherein the nucleic acid has at least 80% identity to SEQ ID NO:1, SEQID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, or SEQ ID NO:23.

The present invention provides in certain embodiments a binding moleculethat binds to TDP-43 associated with ALS and does not bind TDP-43 fromhealthy human brain tissue or TDP-43 associated with FTD, wherein thebinding molecule comprises an amino acid sequence encoded by a nucleicacid, wherein the nucleic acid has at least 80% identity to SEQ ID NO:1,SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ IDNO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11, SEQ IDNO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16, SEQ IDNO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ IDNO:22, or SEQ ID NO:23.

The present invention provides in certain embodiments a method ofbinding TDP-43 associated with ALS comprising contacting a compositionthat comprises TDP-43 associated with ALS with an antibody, antibodyfragment or binding molecule described above.

In certain embodiments, the TDP-43 associated with ALS is in a cell. Incertain embodiments, the TDP-43 associated with ALS is in brain tissue.

The present invention provides in certain embodiments a method ofbinding TDP-43 associated with FTD comprising contacting a compositionthat comprises TDP-43 associated with FTD with an antibody, antibodyfragment or binding molecule described above.

In certain embodiments, the TDP-43 associated with FTD is in a cell. Incertain embodiments, the TDP-43 associated with FTD is in brain tissue.

The present invention provides in certain embodiments a method ofdetecting the presence of TDP-43 associated with FTD in a physiologicalsample comprising contacting a sample with an antibody, antibodyfragment or a binding molecule of any one of claims 1 to 10 anddetermining the binding of the composition with the tissue samplewherein binding of the composition to the tissue sample is indicative ofthe presence of TDP-43 associated with FTD in the tissue sample whereinthe presence of the TDP-43 associated with FTD is indicative offrontotemporal dementia.

The present invention provides in certain embodiments a method ofdetecting the presence of TDP-43 associated with ALS in a physiologicalsample comprising contacting a sample with an antibody, antibodyfragment or a binding molecule of any one of claims 1 to 12 anddetermining the binding of the composition with the tissue samplewherein binding of the composition to the tissue sample is indicative ofthe presence of TDP-43 associated with ALS in the tissue sample whereinthe presence of the TDP-43 associated with ALS is indicative ofamyotrophic lateral sclerosis.

In certain embodiments, the physiological sample is brain tissue, serum,cerebrospinal fluid (CSF), blood, urine or saliva.

The present invention provides in certain embodiments a method oftreating FTD or ALS in the brain of a mammal comprising administering tothe mammal a composition comprising an antibody fragment or a bindingmolecule described above.

The present invention provides in certain embodiments an imagingcomposition specific for TDP-43 associated with ALS or FTD comprising anantibody fragment or a binding molecule described above conjugated to animaging agent.

As used herein, the phrase “specifically recognizes TDP-43” indicatesthat it does not bind to or recognize non-specific proteins.

As used herein, the term “antibody” includes scFv (also called a“nanobody”), humanized, fully human or chimeric antibodies, single-chainantibodies, diabodies, and antigen-binding fragments of antibodies(e.g., Fab fragments). In certain embodiments, the antibody fragmentdoes not contain the constant domain region of an antibody. In certainembodiments, the antibody fragment is less than 500 amino acids inlength, such as between 200-450 amino acids in length, or less than 400amino acids in length.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1B. TDP-43 inclusion morphologies. A) Spinal cord motor neuronswith cytoplasmic skein-like inclusions from patient with sporadic ALS(orig. mag. 400×, anti-TDP43 immunohistochemistry). B) Frontal cortexneuron with nuclear “cat-eye” inclusion from patient with FTD (orig.mag. 1000×).

FIG. 2. Schematic of biopanning protocol. Phage are mixed with targetantigen, deposited on mica, bound phage are attached to target antigenvia antibody binding domain. Unbound phage are removed and bound phagerecovered.

FIG. 3. AFM images of different anti-oligomeric Aβ phage displayednanobodies. C6 binds only to brain derived oligomers, E1 to 1 daysynthetic oligomers and A4 to 3 day synthetic oligomers. Phage appear asfilaments, oligomeric Aβ as small white dots.

FIGS. 4A-4D. 4A. Negative Panning Against Bovine Serum Albumin. 4B.Negative Panning Against Alpha-Synuclein. 4C. Negative Panning AgainstHealthy Homogenized Human Brain Tissue. 4D. Negative Panning AgainstHealthy TDP-43.

FIGS. 5A-5C. Characterization of ALS Clones.

FIG. 6. Characterization of FTD Clones.

FIG. 7. Indirect phage ELISAs using ALT TDP-43 clones and human braintissue homogenates.

FIG. 8. Indirect scFv ELISAs using ALT TDP-43 clones and human braintissue homogenates.

FIG. 9. Dot blot analysis of human brain tissue homogenates using ALSTDP-43 clone 2A.

FIG. 10. Dot blot analysis of ALS TDP-43 scFv production.

FIG. 11. Potential TDP-43 monomeric phages.

FIGS. 12A-C. A. ALS TDP-43 phage capture ELISA with human brain tissue(Monomer phage #3 used for detection). B. ALS TDP-43 phage capture ELISAwith human brain tissue (monomer phage #5 used for detection). C. ALSTDP-43 phage capture ELISA with human brain tissue (commercialanti-TDP43 antibody used for detection).

FIGS. 13A-C. A. ALS TDP-43 phage capture ELISA with IP TDP-43 (monomerphage #3 used for detection). B. ALS TDP-43 phage capture ELISA with IPTDP-43 (monomer phage #5 used for detection). C. ALS TEP-43 phagecapture ELISA with IP TDP-43 (commercial anti-TDP 43 antibody used fordetection).

FIG. 14. scFvs from ALS TDP-43 clones in phage capture ELISA with 4different ALS, FTD and healthy human brain tissue samples.

FIGS. 15A-C. A. Competition ELISA with ALS Capture scFv 2A. B.Competition ELISA with ALS Capture scFv 5E. C. Competition ELISA withALS Capture scFv 8D.

FIGS. 16A-C. Indirect phage ELISA with human plasma samples using ALSTDP-43 clone 5H. B. indirect phage ELISA with human plasma samples usingALS TDP-43 clone 8D. C. Indirect phage ELISA with human plasma samplesusing ALS TDP-43 clone 8E.

FIG. 17. Phage Capture ELISA with FTD and ALS tissue.

FIG. 18. scFv Production of FTD TDP-43 Clones.

DETAILED DESCRIPTION OF THE INVENTION

Protein misfolding and aggregation is a common thread behind manyneurodegenerative diseases including Frontotemporal Degeneration (FTD;also classified as frontotemporal lobar degeneration (FTLD)),Alzheimer's disease (AD), Lewy Body Dementia (LBD), Parkinson's disease(PD), Amyotrophic Lateral Sclerosis (ALS), and Huntington's disease (HD)among others. Numerous studies have implicated small soluble misfoldedoligomeric aggregates of beta-amyloid (Aβ) in the progression of AD andsimilar aggregates of alpha-synuclein (a-syn) in the progression of PDand LBD. Increasing evidence also implicates similar misfoldedoligomeric forms of tau in AD and other tauopathies such as FTD.Misfolding of other proteins including superoxide dismutase andhuntingtin has also been correlated with different neurodegenerativediseases.

Recent evidence has suggested that cytoplasmic misfolding andaggregation of two additional proteins, TAR DNA binding protein 43(TDP-43) and fused in sarcoma protein (FUS) associate with the pathologyobserved in a high percentage of FTD and ALS cases [8]. Thereforeprotein misfolding and aggregation is a key factor behind manyneurodegenerative diseases and reagents which can recognize specificprotein aggregate forms are needed to study the role of these proteinsin disease onset and progression and to facilitate diagnoses. Forexample, as the focus on the role of Aβ in AD slowly shifted towardsoluble Aβ aggregate species instead of insoluble fibrillar aggregates,reagents were needed that could specifically identify the variety ofdifferent aggregate species present. Because of the lack of reagentsthat could distinguish between different Aβ forms, many contradictorystudies on the role of Aβ aggregation in AD were reported and progresson the role of Aβ in AD was significantly impeded. Increasing evidenceindicates that aggregates of TDP-43 play a role in FTD, ALS and otherneurodegenerative diseases including traumatic brain injury. Similar toother neuronal proteins including Aβ, a-syn and tau, TDP-43 is prone toform aggregate species, where TDP-43 mutations linked to increased riskof sporadic ALS aggregate more readily [9]. In order to facilitateidentification of the role of TDP-43 in neurodegenerative disease, wellcharacterized reagents that can specifically recognize the diversity ofTDP-43 morphologies present in the human brain are critically needed tofacilitate studies to identify the most promising TDP-43 forms for useas biomarkers of disease and to study pathogenic/pathophysiologicmechanisms.

While misfolding of certain proteins has been associated with specificdiseases, more than one protein is likely to misfold and aggregate inbrain tissue complicating diagnosis and treatment strategies. Sincecellular stress induced by misfolding and aggregation of one protein maywell lead to misfolding and aggregation of other proteins, the presenceof multiple misfolded proteins in different diseases should be expected.Therefore, there is likely a spectrum of diseases containing variousdifferent aggregated species targeting different cells and regions.Since the various neurodegenerative diseases may have overlappingfeatures, characterizing which aggregated protein species are present atdifferent stages of each disease would greatly facilitate identificationof suitable biomarkers and development of better diagnostic andtreatment strategies. While a number of biomarkers have shown promisefor diagnosing different neurodegenerative diseases (reviewed in [10]),selected aggregate species of Aβ, a-syn and tau have been correlatedwith AD or PD (see for example ([11] [12, 13]) and may have value asselective biomarkers for neurodegenerative disease. It is likely thatdifferent species of TDP-43 can also serve as biomarkers for FTD [14]and other neurodegenerative diseases. TDP-43 is a DNA binding proteinwhich has a number of different alternatively spliced forms [15]. TDP-43binds to a variety of RNA and DNA sequences, particularly to poly-UG RNAsequences [16] accounting for its location in the nucleus, but canshuttle back and forth from the cytoplasm. In FTD and ALS cases,affected neurons and glial cells show a variety of different TDP-43forms accumulate in inclusion bodies in the cytoplasm and/or nucleuswith loss of the normal diffuse nuclear distribution (FIG. 1). TDP-43accumulation occurs in different regions with different types of FTD[17], suggesting that TDP-43 aggregation can be a useful diagnosticbiomarker for these diseases.

Presence of TDP-43 inclusions is also evident in a subset of AD cases,primarily in limbic regions [18, 19] where it can overlap with taupathology [20]. FTD-43 pathology can be induced by increased expressionof Aβ [21] providing additional evidence of the link between keyaggregation prone proteins in the brain including Aβ, tau, a-syn, andTDP-43. There is therefore substantial evidence that aggregation ofTDP-43 is an important factor in FTD, ALS and other neurodegenerativediseases. Aggregation of different protein species, including TDP-43,Aβ, a-syn and tau are important factors in many neurodegenerativediseases and detection of specific aggregate forms of TDP-43 in CSF orserum samples facilitate diagnosis of FTD and provide a means to monitorthe effectiveness of therapeutic strategies. The present inventorsdeveloped novel technology that enables the isolation of single chainantibody fragments, or nanobodies, that selectively bind specificmorphologies of a target protein. Nanobodies have been isolated thatselectively recognize several different oligomeric forms of Aβ andothers against different oligomeric forms of a-syn. The nanobodies canrecognize the various target aggregates in human brain tissue and canreadily distinguish between AD, PD and healthy tissue [1-7].

The present inventors have now developed reagents for detecting specificforms of TDP-43 that are present in FTD and ALS. The nanobodiesspecifically and selectively recognize the most relevant TDP-43aggregate species associated with FTD. The reagents recognizing specificaggregate morphologies of TDP-43 are used in conjunction with otherreagents recognizing specific morphologies of Aβ, a-syn, tau and FUS todistinguish FTD from other neurodegenerative diseases using CSF and/orserum samples.

A vast number of studies have correlated protein aggregation withneurodegenerative diseases including AD, Parkinson's and Dementia withLewy Bodies. Numerous recent studies suggest that specific oligomericforms of these proteins are involved in neuronal toxicity and caninterfere with important functions including long term potentiation.Various soluble oligomeric species of Aβ and a-syn occur early duringthe course of AD and PD, and increasing evidence implicates oligomericforms of tau in AD and other tauopathies.

The inventors developed a novel biopanning technology that combines theimaging capability of Atomic Force Microscopy (AFM) with the diversityof antibody libraries (See FIG. 2). This unique combination of antibodydiversity and imaging capability has enabled the isolation of singlechain antibody variable domain fragment (scFv or nanobody) reagents toan array of morphologies of key proteins involved in neurodegenerativediseases including Aβ and alpha-synuclein (a-syn). Nanobodies wereisolated that specifically recognize monomeric [4], fibrillar [1], andtwo different oligomeric a-syn morphologies [2, 3]. The anti-oligomerica-syn nanobodies do not cross react with oligomeric Aβ, and specificallylabel PD brain tissue but not AD or healthy tissue [3]. In addition,nanobodies were isolated to different regions of full length Aβ [22] andto three distinct naturally occurring oligomeric Aβ morphologies [6,23].

One, A4, specifically recognizes a larger oligomeric Aβ species,inhibits aggregation and extracellular toxicity of Aβ, does not crossreact with oligomeric a-syn, and specifically labels Aβ aggregates inhuman AD brain samples, but not PD or healthy brain tissue [6]. A secondnanobody, E1, recognizes a smaller trimeric or tetrameric Aβ species,and similar to A4 inhibits aggregation and extracellular toxicity of Aβ,does not cross react with oligomeric a-syn, and labels Aβ aggregates inhuman AD but not healthy brain tissue [23]. Utilizing an AD brainderived oligomeric Aβ preparation [24, 25], a third nanobody wasisolated, C6, that specifically recognizes oligomeric Aβ species derivedfrom human AD brain tissue, but does not recognize Aβ aggregatesgenerated in vitro. The different specificities of each nanobody can bereadily observed when each nanobody is expressed on the surface of afilamentous bacteriophage and antibody/antigen complexes are imaged byAFM [23]. Therefore, the combination of antibody libraries and AFMimaging technologies enabled the isolation and characterization ofreagents that recognize specific protein variants including fourdifferent naturally occurring aggregated forms of a-syn and fourdifferent naturally occurring aggregated forms of Aβ.

Another powerful advantage of the present AFM panning protocol is thatnot only can reagents be isolated and characterized to specific proteinmorphologies, but this can be done using only picograms or less ofmaterial. In addition the sample does not need to be purified, and theprotein does not need to be chemically modified in any way. Nanobodieswere actually isolated against a single molecule of the target antigen[26]. This unique combination of capabilities to isolate differentTDP-43 isoforms and to generate and characterize reagents thatspecifically recognize individual protein variants provides the means togenerate reagents that specifically recognize an array of differentTDP-43 variants present in human FTD and ALS brain tissue.

While reagents already exist that can recognize TDP-43, these reagentscannot distinguish between different aggregated states of the protein.Reagents that can detect specific forms of TDP-43 can provide verypowerful tools to facilitate diagnosis of ALS, FTD and otherneurodegenerative diseases, and to follow progression of these diseasesor to evaluate therapeutic strategies. While many neurodegenerativediseases have overlapping clinical symptoms and cellular and biochemicalmechanisms such as an increase in inflammatory markers, and aggregationof similar proteins, the reagents developed by the inventors have welldefined specificities and selectivities for selected TDP-43 forms andfacilitate specific diagnoses of ALS, and FTD and otherneurodegenerative diseases. In combination with other protein andmorphology specific reagents against Aβ and a-syn aggregate species[1-3, 6], these reagents are used to identify disease specificbiomarkers which can readily detect and distinguish many relatedneurodegenerative diseases including ALS, FTD, AD, PD, and LBD.

The present inventors have developed novel technology that enables theisolation of single chain antibody fragments, or nanobodies, thatselectively bind specific morphologies of a target protein. Nanobodieshave been isolated that selectively recognize several differentoligomeric forms of Aβ or a-syn and showed that the nanobodies recognizeaggregates present in diseased but not healthy tissue [1-7].

The inventors have developed selective reagents that can identify whichaggregate morphologies of TDP-43 are specific for diseased human FTD andALS brain issue/biofluids, and which of these TDP-43 species representthe best diagnostic targets. In this proposal we will separate differentaggregate TDP-43 species present in human FTD and ALS brain tissue,generate antibody based reagents (nanobodies) that selectively bind thedifferent TDP-43 aggregate species, and then use the nanobodies toidentify which TDP-43 forms can distinguish between human FTD, ALS andhealthy brain tissue. We will generate nanobodies that recognize humanbrain derived aggregate forms of TDP-43, and utilize these nanobodies toidentify which TDP-43 forms are present in diseased human CSF and braintissue. The hypothesis is that aggregation of TDP-43 is an early eventin FTD and ALS, and that this process can be detected using wellcharacterized nanobodies that selectively recognize specific TDP-43aggregate forms.

To achieve the goal of developing nanobodies that specifically andselectively recognize the most relevant TDP-43 aggregate speciesassociated with FTD and ALS, the inventors isolated different sizeoligomeric TDP-43 species from human FTD and ALS brain tissue, generatedand characterized nanobodies against specific brain derived TDP-43 formsisolated from FTD and ALS brain tissue, and identified TDP-43 morphologyspecific nanobodies that distinguish between brain tissue from FTD, ALSand healthy human post-mortem cases.

The inventors generated nanobody reagents that selectively recognizedvariants of TDP-43 that are associated with FTD or ALS. Identificationof TDP-43 forms that can be used for diagnosis and staging of FTD andALS and potentially other neurodegenerative diseases provide valuablebiomarkers for disease diagnosis and progression, and identify potentialtherapeutic targets. The morphology specific nanobodies developed hereserve as valuable reagents in various biosensor formats to detectspecific TDP-43 morphologies for diagnostic tests or to follow diseaseprogression or as reagents to specifically target toxic TDP-43 speciesfor intracellular and extracellular therapeutic applications. Whilereagents already exist that can recognize TDP-43, the prior reagentscannot distinguish between different aggregated states of the protein.The nanobody reagents presently developed are used either extra- orintracellularly to identify or target specific TDP-43 morphologiesinvolved in the onset and progression of ALS and FTD. The reagents arealso used in conjunction with other reagents recognizing specificmorphologies of Aβ, a-syn and tau to characterize the concentrationprofiles of these markers in CSF and/or serum samples from patients withvarious neurodegenerative diseases and to monitor the progression ofthese diseases.

In the present invention, using a bio-panning protocol to identifysingle chain antibody fragments (scFv, also called nanobodies) againstlow (pico-molar) quantities of specific TDP-43 morphologies, theinventors identified binding reagents with therapeutic and diagnosticproperties. Specifically, the inventors have generated single chainantibody fragments (scFvs or nanobodies) that selectively recognizespecific morphologies of the protein TDP-43. These isolated scFvs thathave potential value as diagnostics, therapeutics and imaging agents forneurodegeneration. As diagnostics, these antibody fragments can be usedto detect the presence of specific morphologies of the protein TDP-43 inserum, CSF or other fluid samples as a presymptomatic indication ofneurodegeneration. Specific morphologies of the protein TDP-43 may be anindicator of particular neurodegenerative diseases. The antibodyfragments can also be used as therapeutics to selectively target thespecific morphologies of the protein TDP-43. Finally, the reagents canalso be used as imaging agents to detect the presence of specificmorphologies of the protein TDP-43 in vivo. The antibody fragments canbe readily labeled for PET scans or other imaging techniques.

The biopanning studies were performed to isolate single chain variablefragments (nanobodies) against the different TDP-43 species. Thebiopanning protocol that was used combines the imaging capabilities ofAFM with the binding diversity of phage-displayed antibody technology.

In a broad sense the scFv compositions of the present invention may bedescribed as compounds that are binding compounds specific for targetmorphologies of TDP-43. These compounds may therefore be used indiagnostic as well therapeutic applications and may be eitheradministered to patients or used on patient tissue samples. In someembodiments, the compositions of the present invention may be used forin vivo imaging of target morphologies of TDP-43, and distinguishbetween neurological tissue with forms of TDP-43 associated with ALS orFTD, and TDP-43 associated with normal neurological tissue. As such thenanobody compositions of the invention may be used to detect andquantitate TDP-43 in diseases including, for example, ALS andfrontotemporal dementia. In another embodiment, the compounds may beused in the treatment or prophylaxis of neurodegenerative disorders.Also provided herein are methods of allowing the compound to distributeinto the brain tissue, and imaging the brain tissue, wherein an increasein binding of the compound to the brain tissue compared to a normalcontrol level of binding indicates that the mammal is suffering from oris at risk of developing a neurodegenerative disease, such as ALS orfrontotemporal dementia.

The methods of the present invention are conducted to provide earlystage diagnosis of ALS and FTD. As explained herein the nanobodies ofthe invention are ones that specifically recognize TDP-43 associatedwith ALS or FTD. Thus, compositions comprising these antibodies andantibody fragments may be used to identify the presence of TDP-43associated with ALS or FTD in a biological sample from a patient to betested for a neurodegenerative disease, wherein the presence of TDP-43associated with ALS or FTD in the sample is indicative that the patienthas or is likely to develop ALS or FTD. In certain embodiments, theassay format that is used may be any assay format that typically employsantibody compositions. Thus, for example, the biological sample may beexamined using immunohistology techniques, ELISA, Western Blotting, andthe like.

For purposes of the diagnostic methods of the invention, thecompositions of the invention may be conjugated to a detecting reagentthat facilitates detection of the scFv. For example, example, thedetecting reagent may be a direct label or an indirect label. The labelscan be directly attached to or incorporated into the detection reagentby chemical or recombinant methods.

In one embodiment, a label is coupled to the scFv through a chemicallinker. Linker domains are typically polypeptide sequences, such as polygly sequences of between about 5 and 200 amino acids. In someembodiments, proline residues are incorporated into the linker toprevent the formation of significant secondary structural elements bythe linker. In certain embodiments, linkers are flexible amino acidsubsequences that are synthesized as part of a recombinant fusionprotein comprising the RNA recognition domain. In one embodiment, theflexible linker is an amino acid subsequence that includes a proline,such as Gly(x)-Pro-Gly(x) where x is a number between about 3 and about100. In other embodiments, a chemical linker is used to connectsynthetically or recombinantly produced recognition and labeling domainsubsequences. Such flexible linkers are known to persons of skill in theart. For example, poly(ethylene glycol) linkers are available fromShearwater Polymers, Inc. Huntsville, Ala. These linkers optionally haveamide linkages, sulthydryl linkages, or heterofunctional linkages.

The detectable labels can be used in the assays of the present inventionto diagnose a neurodegenerative disease, such as ALS or FTD, theselabels are attached to the scFvs of the invention, can be primary labels(where the label comprises an element that is detected directly or thatproduces a directly detectable element) or secondary labels (where thedetected label binds to a primary label, e.g., as is common inimmunological labeling). An introduction to labels, labeling proceduresand detection of labels is found in Polak and Van Noorden (1997)Introduction to Immunocytochemistry, 2nd ed., Springer Verlag, N.Y. andin Haugland (1996) Handbook of Fluorescent Probes and ResearchChemicals, a combined handbook and catalogue Published by MolecularProbes, Inc., Eugene, Oreg. Patents that described the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149; and 4,366,241.

Primary and secondary labels can include undetected elements as well asdetected elements. Useful primary and secondary labels in the presentinvention can include spectral labels such as green fluorescent protein,fluorescent dyes (e.g., fluorescein and derivatives such as fluoresceinisothiocyanate (FITC) and Oregon Green™, rhodamine and derivatives(e.g., Texas red, tetrarhodimine isothiocynate (TRITC), etc.),digoxigenin, biotin, phycoerythrin, AMCA, CyDyes™, and the like),radiolabels (e.g., ³H, ¹²⁵I, ³⁵S, ¹⁴C, ³²P, ³³P, etc.), enzymes (e.g.,horse radish peroxidase, alkaline phosphatase etc.), spectralcalorimetric labels such as colloidal gold or colored glass or plastic(e.g. polystyrene, polypropylene, latex, etc.) beads. The label can becoupled directly or indirectly to a component of the detection assay(e.g., the detection reagent) according to methods well known in theart. As indicated above, a wide variety of labels may be used, with thechoice of label depending on sensitivity required, ease of conjugationwith the compound, stability requirements, available instrumentation,and disposal provisions.

Exemplary labels that can be used include those that use: 1)chemiluminescence (using horseradish peroxidase and/or alkalinephosphatase with substrates that produce photons as breakdown productsas described above) with kits being available, e.g., from MolecularProbes, Amersham, Boehringer-Mannheim, and Life Technologies/Gibco BRL;2) color production (using both horseradish peroxidase and/or alkalinephosphatase with substrates that produce a colored precipitate (kitsavailable from Life Technologies/Gibco BRL, and Boehringer-Mannheim));3) fluorescence using, e.g., an enzyme such as alkaline phosphatase,together with the substrate AttoPhos (Amersham) or other substrates thatproduce fluorescent products, 4) fluorescence (e.g., using Cy-5(Amersham), fluorescein, and other fluorescent tags); 5) radioactivity.Other methods for labeling and detection will be readily apparent to oneskilled in the art.

Where the scFv-based compositions of the invention are contemplated tobe used in a clinical setting, the labels are preferably non-radioactiveand readily detected without the necessity of sophisticatedinstrumentation. In certain embodiments, detection of the labels willyield a visible signal that is immediately discernable upon visualinspection. One example of detectable secondary labeling strategies usesan antibody that recognizes TDP-43 associated with ALS or FTD in whichthe antibody is linked to an enzyme (typically by recombinant orcovalent chemical bonding). The antibody is detected when the enzymereacts with its substrate, producing a detectable product. In certainembodiments, enzymes that can be conjugated to detection reagents of theinvention include, e.g., β-galactosidase, luciferase, horse radishperoxidase, and alkaline phosphatase. The chemiluminescent substrate forluciferase is luciferin. One embodiment of a fluorescent substrate forβ-galactosidase is 4-methylumbelliferyl-β-D-galactoside. Embodiments ofalkaline phosphatase substrates include p-nitrophenyl phosphate (pNPP),which is detected with a spectrophotometer; 5-bromo-4-chloro-3-indolylphosphate/nitro blue tetrazolium (BCIP/NBT) and fast red/napthol AS-TRphosphate, which are detected visually; and4-methoxy-4-(3-phosphonophenyl) spiro[1,2-dioxetane-3,2′-adamantane],which is detected with a luminometer. Embodiments of horse radishperoxidase substrates include 2,2′azino-bis(3-ethylbenzthiazoline-6sulfonic acid) (ABTS), 5-aminosalicylic acid (5AS), o-dianisidine, ando-phenylenediamine (OPD), which are detected with a spectrophotometer,and 3,3,5,5′-tetramethylbenzidine (TMB), 3,3′ diaminobenzidine (DAB),3-amino-9-ethylcarbazole (AEC), and 4-chloro-1-naphthol (4C1N), whichare detected visually. Other suitable substrates are known to thoseskilled in the art. The enzyme-substrate reaction and product detectionare performed according to standard procedures known to those skilled inthe art and kits for performing enzyme immunoassays are available asdescribed above.

The presence of a label can be detected by inspection, or a detectorwhich monitors a particular probe or probe combination is used to detectthe detection reagent label. Typical detectors includespectrophotometers, phototubes and photodiodes, microscopes,scintillation counters, cameras, film and the like, as well ascombinations thereof. Examples of suitable detectors are widelyavailable from a variety of commercial sources known to persons ofskill. Commonly, an optical image of a substrate comprising boundlabeling moieties is digitized for subsequent computer analysis.

As noted herein throughout the scFvs of the invention are targetedspecifically to TDP-43 associated with ALS or FTD. As such, the scFvs ofthe invention also may be used to specifically target therapeuticcompositions to the sites of aggregation of TDP-43 associated with ALSor FTD. In this embodiment, any therapeutic agent typically used for thetreatment of these diseases, may be conjugated to scFvs in order toachieve a targeted delivery of that therapeutic agent.

The scFv compositions of the invention can be used in any diagnosticassay format to determine the presence of TDP-43 associated with ALS orFTD. A variety of immunodetection methods are contemplated for thisembodiment. Such immunodetection methods include enzyme linkedimmunosorbent assay (ELISA), radioimmunoassay (RIA), immunoradiometricassay, fluoroimmunoassay, chemiluminescent assay, bioluminescent assay,and Western blot, though several others are well known to those ofordinary skill. The steps of various useful immunodetection methods havebeen described in the scientific literature.

In general, the immunobinding methods include obtaining a samplesuspected of containing a protein, polypeptide and/or peptide (in thiscase TDP-43 associated with ALS or FTD), and contacting the sample witha first antibody, monoclonal or polyclonal, in accordance with thepresent invention, as the case may be, under conditions effective toallow the formation of immunocomplexes.

The immunobinding methods include methods for detecting and quantifyingthe amount of the TDP-43 associated with ALS or FTD in a sample and thedetection and quantification of any immune complexes formed during thebinding process. Here, one would obtain a sample suspected of containingTDP-43 associated with ALS or FTD, and contact the sample with anantibody fragment of the invention, and then detect and quantify theamount of immune complexes formed under the specific conditions.

Contacting the chosen biological sample with the antibody undereffective conditions and for a period of time sufficient to allow theformation of immune complexes (primary immune complexes) is generally amatter of simply adding the antibody composition to the sample andincubating the mixture for a period of time long enough for theantibodies to form immune complexes with, i.e., to bind to, any antigenspresent. After this time, the sample-antibody composition, such as atissue section, ELISA plate, dot blot or western blot, will generally bewashed to remove any non-specifically bound antibody species, allowingonly those scFv molecules specifically bound within the primary immunecomplexes to be detected.

In general, the detection of immunocomplex formation is well known inthe art and may be achieved through the application of numerousapproaches. These methods are generally based upon the detection of alabel or marker, such as any of those radioactive, fluorescent,biological and enzymatic tags. U.S. patents concerning the use of suchlabels include U.S. Pat. Nos. 3,817,837; 3,850,752; 3,939,350;3,996,345; 4,277,437; 4,275,149 and 4,366,241, each incorporated hereinby reference. Of course, one may find additional advantages through theuse of a secondary binding ligand such as a second antibody and/or abiotin/avidin ligand binding arrangement, as is known in the art.

As noted above, an scFv of the invention may itself be linked to adetectable label, wherein one would then simply detect this label,thereby allowing the amount of the primary immune complexes in thecomposition to be determined. Alternatively, the first antibody thatbecomes bound within the primary immune complexes may be detected bymeans of a second binding ligand that has binding affinity for theantibody. In these cases, the second binding ligand may be linked to adetectable label. The second binding ligand is itself often an antibody,which may thus be termed a “secondary” antibody. The primary immunecomplexes are contacted with the labeled, secondary binding ligand, orantibody, under effective conditions and for a period of time sufficientto allow the formation of secondary immune complexes. The secondaryimmune complexes are then generally washed to remove anynon-specifically bound labeled secondary antibodies or ligands, and theremaining label in the secondary immune complexes is then detected.

Further methods include the detection of primary immune complexes by atwo step approach. A second binding ligand, such as an antibody, thathas binding affinity for the scFV is used to form secondary immunecomplexes, as described above. After washing, the secondary immunecomplexes are contacted with a third binding ligand or antibody that hasbinding affinity for the second antibody, again under effectiveconditions and for a period of time sufficient to allow the formation ofimmune complexes (tertiary immune complexes). The third ligand orantibody is linked to a detectable label, allowing detection of thetertiary immune complexes thus formed. This system may provide forsignal amplification if this is desired.

One method of immunodetection uses two different antibodies. A firststep biotinylated, monoclonal or polyclonal antibody (in the presentexample a scFv of the invention) is used to detect the targetantigen(s), and a second step antibody is then used to detect the biotinattached to the complexed nanobody. In this method the sample to betested is first incubated in a solution containing the first stepnanobody. If the target antigen is present, some of the nanobody bindsto the antigen to form a biotinylated nanobody/antigen complex. Thenanobody/antigen complex is then amplified by incubation in successivesolutions of streptavidin (or avidin), biotinylated DNA, and/orcomplementary biotinylated DNA, with each step adding additional biotinsites to the nanobody/antigen complex. The amplification steps arerepeated until a suitable level of amplification is achieved, at whichpoint the sample is incubated in a solution containing the second stepantibody against biotin. This second step antibody is labeled, as forexample with an enzyme that can be used to detect the presence of theantibody/antigen complex by histoenzymology using a chromogen substrate.With suitable amplification, a conjugate can be produced which ismacroscopically visible.

Another known method of immunodetection takes advantage of theimmuno-PCR (Polymerase Chain Reaction) methodology. The PCR method issimilar to the method described above up to the incubation withbiotinylated DNA. However, instead of using multiple rounds ofstreptavidin and biotinylated DNA incubation, theDNA/biotin/streptavidin/antibody complex is washed out with a low pH orhigh salt buffer that releases the antibody. The resulting wash solutionis then used to carry out a PCR reaction with suitable primers withappropriate controls. At least in theory, the enormous amplificationcapability and specificity of PCR can be utilized to detect a singleantigen molecule.

As detailed above, immunoassays, in their most simple and/or directsense, are binding assays. Certain preferred immunoassays are thevarious types of enzyme linked immunosorbent assays (ELISAs) and/orradioimmunoassays (RIA) known in the art. Immunohistochemical detectionusing tissue sections is also particularly useful. However, it will bereadily appreciated that detection is not limited to such techniques,and/or western blotting, dot blotting, FACS analyses, and/or the likemay also be used.

The diagnostic assay format that may be used in the present inventioncould take any conventional format such as ELISA or other platforms suchas luminex or biosensors. The present invention shows the sequence ofcertain exemplary DNA sequences for binding agents specific for TDP-43associated with ALS or FTD. These sequences can readily be modified tofacilitate diagnostic assays, for example a tag (such as GFP) can beadded to these scFvs to increase sensitivity. In one exemplary ELISA,antibodies are immobilized onto a selected surface exhibiting proteinaffinity, such as a well in a polystyrene microtiter plate. Then, a testcomposition suspected of containing TDP-43 associated with ALS or FTD,such as a clinical sample (e.g., a biological sample obtained from thesubject), is added to the wells. After binding and/or washing to removenon-specifically bound immune complexes, the bound antigen may bedetected. Detection is generally achieved by the addition of anotherantibody that is linked to a detectable label. This type of ELISA is asimple “sandwich ELISA.” Detection may also be achieved by the additionof a second antibody, followed by the addition of a third antibody thathas binding affinity for the second antibody, with the third antibodybeing linked to a detectable label.

In another exemplary ELISA, the samples suspected of containing theantigen are immobilized onto the well surface and/or then contacted withbinding agents (e.g., scFvs of the invention). After binding and/orwashing to remove non-specifically bound immune complexes, the boundanti-binding agents are detected. Where the initial binding agents arelinked to a detectable label, the immune complexes may be detecteddirectly. Again, the immune complexes may be detected using a secondantibody that has binding affinity for the first binding agents, withthe second antibody being linked to a detectable label.

Another ELISA in which the antigens are immobilized, involves the use ofantibody competition in the detection. In this ELISA, labeled antibodies(or nanobodies) against an antigen are added to the wells, allowed tobind, and/or detected by means of their label. The amount of an antigenin an unknown sample is then determined by mixing the sample with thelabeled antibodies against the antigen during incubation with coatedwells. The presence of an antigen in the sample acts to reduce theamount of antibody against the antigen available for binding to the welland thus reduces the ultimate signal. This is also appropriate fordetecting antibodies against an antigen in an unknown sample, where theunlabeled antibodies bind to the antigen-coated wells and also reducesthe amount of antigen available to bind the labeled antibodies.

Irrespective of the format employed, ELISAs have certain features incommon, such as coating, incubating and binding, washing to removenon-specifically bound species, and detecting the bound immunecomplexes.

In coating a plate with either TDP-43 associated with ALS or FTD or anscFv of the invention, one will generally incubate the wells of theplate with a solution of the antigen or scFvs, either overnight or for aspecified period of hours. The wells of the plate will then be washed toremove incompletely adsorbed material. Any remaining available surfacesof the wells are then “coated” with a nonspecific protein that isantigenically neutral with regard to the test antisera. These includebovine serum albumin (BSA), casein or solutions of milk powder. Thecoating allows for blocking of nonspecific adsorption sites on theimmobilizing surface and thus reduces the background caused bynonspecific binding of antisera onto the surface.

In ELISAs, it is probably more customary to use a secondary or tertiarydetection means rather than a direct procedure. Thus, after binding of aprotein or antibody to the well, coating with a non-reactive material toreduce background, and washing to remove unbound material, theimmobilizing surface is contacted with the biological sample to betested under conditions effective to allow immune complex(antigen/antibody) formation. Detection of the immune complex thenrequires a labeled secondary binding ligand or antibody, and a secondarybinding ligand or antibody in conjunction with a labeled tertiaryantibody or a third binding ligand.

“Under conditions effective to allow immune complex (antigen/antibody)formation” means that the conditions preferably include diluting theTDP-43 associated with ALS or FTD and/or scFv composition with solutionssuch as BSA, bovine gamma globulin (BGG) or phosphate buffered saline(PBS)/Tween. These added agents also tend to assist in the reduction ofnonspecific background.

The “suitable” conditions also mean that the incubation is at atemperature or for a period of time sufficient to allow effectivebinding. Incubation steps are typically from about 1 to 2 to 4 hours orso, at temperatures preferably on the order of 25° C. to 27° C., or maybe overnight at about 4° C. or so.

Following all incubation steps in an ELISA, the contacted surface iswashed so as to remove non-complexed material. An example of a washingprocedure includes washing with a solution such as PBS/Tween, or boratebuffer. Following the formation of specific immune complexes between thetest sample and the originally bound material, and subsequent washing,the occurrence of even minute amounts of immune complexes may bedetermined.

To provide a detecting means, the second or third antibody will have anassociated label to allow detection. This may be an enzyme that willgenerate color development upon incubating with an appropriatechromogenic substrate. Thus, for example, one will desire to contact orincubate the first and second immune complex with a urease, glucoseoxidase, alkaline phosphatase or hydrogen peroxidase-conjugated antibodyfor a period of time and under conditions that favor the development offurther immune complex formation (e.g., incubation for 2 hours at roomtemperature in a PBS-containing solution such as PBS-Tween).

After incubation with the labeled antibody, and subsequent to washing toremove unbound material, the amount of label is quantified, e.g., byincubation with a chromogenic substrate such as urea, or bromocresolpurple, or 2,2′-azino-di-(3-ethyl-benzthiazoline-6-sulfonic acid (ABTS),or H₂O₂, in the case of peroxidase as the enzyme label. Quantificationis then achieved by measuring the degree of color generated, e.g., usinga visible spectra spectrophotometer.

In various aspects of the invention, it will be desirable to furthersubject patients to more traditional diagnostic approaches for ALS orFTD.

As noted above, there are various drugs that are presently in use orunder development for the treatment of ALS and frontotemporal dementia.The present invention contemplates the use of scFvs of the invention,based “diagnostic” methods to further assess the efficacy of treatments.Given the role of TDP-43 associated with ALS or FTD in these diseases,the ability of a particular therapy to reduce the amount of TDP-43associated with ALS or FTD will be indicative of an effective treatment.

The present invention may involve the use of pharmaceutical compositionswhich comprise an agent conjugated to a scFv of the invention fordelivery into a subject having ALS or frontotemporal dementia. Such anagent will ideally be formulated into a pharmaceutically acceptablecarrier. As used herein, “pharmaceutically acceptable carrier” includesany and all solvents, dispersion media, coatings, surfactants,antioxidants, preservatives (e.g., antibacterial agents, antifungalagents), isotonic agents, absorption delaying agents, salts,preservatives, drugs, drug stabilizers, gels, binders, excipients,disintegration agents, lubricants, sweetening agents, flavoring agents,dyes, such like materials and combinations thereof, as would be known toone of ordinary skill in the art. Except insofar as any conventionalcarrier is incompatible with the active ingredient, its use in thetherapeutic or pharmaceutical compositions is contemplated.

An amino acid sequence of an antibody or antibody fragment or variantthereof described herein or a nucleic acid sequence or variant thereofencoding such an amino acid sequence, is a sequence that issubstantially similar to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ IDNO:4, SEQ ID NO:5, or SEQ ID NO:6. Variant amino acid and nucleic acidsequences include synthetically derived amino acid and nucleic acidsequences, or recombinantly derived amino acid or nucleic acidsequences. Generally, nucleic acid or amino acid sequences of theinvention will have at least 40 to 100% sequence identity to SEQ IDNO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, or SEQ IDNO:6. In certain embodiments, the nucleic acid or amino acid sequencesof the invention will have at least 50, 60, 70%, 71%, 72%, 73%, 74%,75%, 76%, 77%, 78% 79%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequenceidentity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ IDNO:5, or SEQ ID NO:6.

“Variants” are intended to include sequences derived by deletion(so-called truncation) or addition of one or more amino acids to theN-terminal and/or C-terminal end, and/or addition of one or more basesto the 5′ or 3′ end of the nucleic acid sequence; deletion or additionof one or more amino acids/nucleic acids at one or more sites in thesequence; or substitution of one or more amino acids/nucleic acids atone or more sites in the sequence. The antibodies and antibody fragmentsdescribed herein may be altered in various ways including amino acidsubstitutions, deletions, truncations, and insertions. Methods for suchmanipulations are generally known in the art. For example, amino acidsequence variants of the enzyme can be prepared by mutations in the DNA.Methods for mutagenesis and nucleotide sequence alterations are wellknown in the art. The substitution may be a conserved substitution. A“conserved substitution” is a substitution of an amino acid with anotheramino acid having a similar side chain. A conserved substitution wouldbe a substitution with an amino acid that makes the smallest changepossible in the charge of the amino acid or size of the side chain ofthe amino acid (alternatively, in the size, charge or kind of chemicalgroup within the side chain) such that the overall enzyme retains itsspatial conformation but has altered biological activity. For example,common conserved changes might be Asp to Glu, Asn or Gln; His to Lys,Arg or Phe; Asn to Gln, Asp or Glu and Ser to Cys, Thr or Gly. Alanineis commonly used to substitute for other amino acids. The 20 essentialamino acids can be grouped as follows: alanine, valine, leucine,isoleucine, proline, phenylalanine, tryptophan and methionine havingnonpolar side chains; glycine, serine, threonine, cystine, tyrosine,asparagine and glutamine having uncharged polar side chains; aspartateand glutamate having acidic side chains; and lysine, arginine, andhistidine having basic side chains.

As used herein, “sequence identity” or “identity” in the context of twonucleic acid or polypeptide sequences makes reference to a specifiedpercentage of residues in the two sequences that are the same whenaligned for maximum correspondence over a specified comparison window,as measured by sequence comparison algorithms or by visual inspection.When percentage of sequence identity is used in reference to proteins itis recognized that residue positions which are not identical oftendiffer by conservative amino acid substitutions, where amino acidresidues are substituted for other amino acid residues with similarchemical properties (e.g., charge or hydrophobicity) and therefore donot change the functional properties of the molecule. When sequencesdiffer in conservative substitutions, the percent sequence identity maybe adjusted upwards to correct for the conservative nature of thesubstitution. Sequences that differ by such conservative substitutionsare said to have “sequence similarity” or “similarity.” Means for makingthis adjustment are well known to those of skill in the art. Typicallythis involves scoring a conservative substitution as a partial ratherthan a full mismatch, thereby increasing the percentage sequenceidentity. Thus, for example, where an identical amino acid is given ascore of 1 and a non-conservative substitution is given a score of zero,a conservative substitution is given a score between zero and 1. Thescoring of conservative substitutions is calculated, e.g., asimplemented in the program PC/GENE (Intelligenetics, Mountain View,Calif.).

EXAMPLES Example 1 Preparation of Brain Derived TDP-43 Aggregates

TDP-43's role in normal cell biology and in neuropathological processesin not well understood. Aberrant localization in the cytoplasm or innuclear aggregates is seen in regionally specific glia and neurons inboth sporadic and some familial forms of ALS and FTDs [8, 31]. Severalreports have looked at TDP-43 as a biomarker and measured elevated CSFand plasma levels in some subsets of patients with FTD and ALS, thoughsensitivity and specificity has been a problem (reviewed in [32, 33]).Appropriate normal and disease cases were identified and TDP-43aggregates were purified by immunoprecipitation. Next, nanobodies weregenerated against the different TDP-43 variants. Third, the nanobodieswere used to identify which forms of TDP-43 best distinguished ALS, FTDand healthy brain tissue.

Purification and Characterization of Oligomeric TDP-43.

The different monomeric and aggregated species of TDP-43 were purifiedfrom normal brain, FTLD-TDP [34], sporadic ALS, and ALS-FTD brainspecimens. Samples were chosen from affected regions of frontal ortemporal cortex and confirmed to have abundant numbers of TDP-43aggregates by standard immunohistochemistry in adjacent FFPE tissues. Asimple semiquantitative scoring scheme for TDP-43 aggregate burden intissue has recently been reported in an interesting case report [31] andwas recorded for each sample. Recording of types of inclusions and cellsaffected was also be done (i.e., cytoplasmic, nuclear, neuronal, glial).Post-mortem samples have been deidentified for all personal patientinformation and have confirmed neuropathological diagnoses and reportsavailable. Samples were chosen for post-mortem intervals less than 24hours (optimally less than 12 hours). Brain tissue was maintained at−80° C. until use.

Three to five cases for each group were selected, andimmunoprecipitation for TDP-43 samples from each group was preparedusing standard methods as described recently [35]. Briefly, frozencortical samples were homogenized in cold lysis buffer: 25 mM HEPES-NaOH(pH 7.9), 150 mM NaCl, 1.5 mM MgCl₂, 0.2 mM EDTA, 0.5% Triton-X-100, 1mM dithiothreitol, protease inhibitor cocktail. The lysate was incubatedwith 50 μl of Dynabeads (Protein-G beads, Invitrogen) according tomanufacturer protocol. In the first stage, beads were coated withanti-TDP-43 polyclonal (ProteinTech, Chicago, Ill., USA). Aftersubsequent washing, the beads were incubated overnight at 4° C. with 400mg of cell lysate. The beads were rinsed twice with PBS andantibody-bound complexes are eluted by boiling in SDS loading buffer(0.5 mM Tris-HCl pH: 6.8; 10% SDS; 5% Glycerol). Samples were frozendown. Proteins were also resolved by 10% SDS-PAGE and transferred onnitrocellulose membrane (Biorad, Hercules, Calif., USA). The membranewas incubated with anti-TDP-43 antibody, and immunoreactive proteinswere visualized by chemilluminescence (Perkin and Elmer, Santa Clara,Calif., USA).

Generation and Characterization of Nanobodies Against Specific BrainDerived TDP-43 Forms Isolated from FTD and ALS Brain Tissue.

Protocols have been developed that enable the ready isolation ofindividual clones from phage display libraries that recognize specificprotein morphologies. Panning protocols have continued to be refined tofacilitate isolation of reagents against targets that are available inlimited amounts, that cannot be purified or that are unstable. Resultshave been reported for the isolation of nanobodies against monomeric,fibrillar and two different oligomeric forms of a-syn, and monomeric,fibrillar and two different oligomeric β-amyloid species using the basicprotocol illustrated in FIG. 2 [1-4, 6, 7, 22, 23, 36]. The isolation ofa nanobody against a third distinct oligomeric beta-amyloid morphologyhas also been accomplished. The inventors have now included additionalnegative panning steps to remove nonspecific and undesired bindingactivities, so virtually all clones isolated after only a single roundof panning specifically recognize the target antigen. Using thistechnique, the inventors were able to isolate various morphologyspecific ligands using only nanograms of target. The inventors have alsodeveloped AFM based protocols to characterize ligand binding [37], sonot only were they able to isolate morphology specific ligands with onlyminimal material, but they were able to characterize binding specificitywith limited material as well. This unique capability is ideally suitedto isolating ligands against specific protein morphologies.

AFM Panning Against TDP-43 Aggregates.

Similar protocols were used to isolate nanobodies to the brain derivedTDP-43 aggregate morphologies as described previously [1-3, 6]. Onlynanogram amounts of material was needed for the panning protocols, sominimal amounts of material were needed for the panning process. Toensure that the nanobodies isolated from the panning protocolselectively recognized FTD or ALS brain derived TDP-43 aggregatespecies, a series of negative panning steps were performed prior to thepositive selection on the brain derived samples. First, a negativepanning step was utilized on the control protein BSA to remove allnon-specific sticky nanobodies. Next, a negative panning step usingcontrol non-diseased brain sample that was prepared similarly to thedisease brain samples was used to remove nanobodies binding tonon-disease associated forms of TDP-43 and any brain proteins that maypurify with TDP-43. Finally, a third negative panning step usingsynthetic monomeric TDP-43 (Proteintech) was used to remove allnanobodies against monomeric TDP-43. AFM was utilized to verify that allphage binding to the non-target samples have been removed for eachnegative panning step. An aliquot of the remaining phage was added tomica containing an aliquot of the non-target sample and unbound phagewere removed. If any phage were observed still binding to the off targetsamples, a second round of negative panning was performed. The processwas repeated until no remaining phage bind the off target sample. Afterthe negative panning steps, the remaining phage were added to an aliquotof either the positive FTD or ALS brain derived TDP-43 aggregate sampleand positive clones recovered as described [1-3, 6, 23, 38, 39].

Nanobody Characterization.

There are numerous techniques that can be used to determine bindingspecificity of each of the nanobodies isolated against the differenttarget TDP-43 morphologies depending on the availability and stabilityof the target antigen. In characterizing binding specificity, many ofthe same problems that were encountered in biopanning arise again, theprimary one being that it is difficult to determine nanobody bindingspecificity to aggregate morphologies that are present in small amounts,that may be difficult to purify or that are not amenable to standardimmobilization protocols. To address these problems, the inventorsutilized the capabilities of AFM to develop new techniques that enabledthe characterization of binding specificity of nanobodies to differenttarget protein morphologies using minimal material and without the needfor any modification. Therefore it was possible to employ a variety ofdifferent binding assays depending on how much of the target proteinmorphology can be purified, whether it can be purified to homogeneity ornot, and how stable the particular target morphology was. The results wehave obtained with the various assays are described.

Specificity Using Biacore, ELISA, Western Blot or Dot Blot.

For those TDP-43 morphologies that were obtained in reasonable quantity,it was possible to determine accurate binding kinetics by surfaceplasmon resonance using a BIAcore X biosensor. Since chemicalimmobilization may affect various aggregated protein morphologies, it ispossible to determine binding specificity by ELISA, western or dot blot,depending on how easy it is to purify the target aggregate morphology.The protocols for each of these assays are routinely used [2, 4, 7, 22,36, 40, 41].

Specificity Using AFM.

For certain of the aggregated TDP-43 samples, it is not possible todetermine nanobody specificity by conventional means such as westernblot as described above. Several different AFM based methods have beendeveloped and used to determine antibody specificity for antigen targetsthat are not suitable for analysis as described above, or that areavailable in only limited amounts.

In certain situations nanobody specificity can be identified by heightdistribution analysis as described [37]. In this AFM based assay, theantigen is deposited on the AFM substrate and the height distribution ofthe sample is analyzed. The nanobody is then added to the immobilizedtarget antigen and the height distribution reanalyzed. If the nanobodybinds to the target antigen, the particle height changes, if not itremains constant. It can be determined which specific particle heightsare interacting with each different nanobody using this technique asdescribed [37]. It can also be determined the extent of nanobody bindingas a function of nanobody concentration, generating a Langmuir isothermplot from which we can calculate a KD value for the nanobody. It canalso be determined using a technique termed recognition imaging [42],where the nanobody is chemically fixed to a short tether on the AFM tip.The antigen sample is added to the AFM substrate and imaged using thetethered AFM tip. The resulting scan provides the standard AFM imagealong with an overlay showing specific locations where the nanobody hasdeflected the tether because it is binding to antigen on the scannedsurface. Using this technique it was possible to show that a nanobodyagainst fibrillar Aβ binds at fixed repetitive intervals to fibrillar Aβindicative of the helical structure of the fibrils [42]. Finally it ispossible to directly image whether a phage displayed version of thenanobody binds to a given target. The nanobodies are displayed on thesurface of bacteriophage for easy visualization by AFM. Using thistechnique the inventors showed that three different nanobodies againstoligomeric Aβ recognize three distinctly different oligomeric Aβ species(see FIG. 3). All positive nanobodies were screened for selectivitytowards FTD, ALS and healthy brain samples.

Identification of TDP-43 Morphology Specific Nanobodies that DistinguishBetween Brain Tissue from FTD, ALS and Healthy Human Post-Mortem Cases.

Individual nanobodies were screened against normal, FTLD-TDP andsporadic ALS brain tissue specimens using ELISA, dot blot andimmunohistochemistry to identify those nanobody reagents that have themost potential to distinguish between healthy and disease cases.

Western- and Dot-Blot Assays:

All assays were performed essentially as described [2, 3].

Immunohistochemistry of Brain Tissue.

Formalin-fixed paraffin-embedded brain tissue sections from wellcharacterized normal and neurodegenerative cases are pre-treated with0.1% triton X-100 for 30 minutes. Nanobody is then added (0.2 mg/ml) tothe brain sections and incubated for 1 hour at room temperature. Primaryantibodies (mouse anti-c-Myc to label nanobodies containing a c-myc tag,and neuronal markers such as anti-Synaptophysin (Santa Cruz) 1/500dilution in BSA 3%) are then applied and incubated for 1 hour at roomtemperature. The brain sections are washed 3 times with PBS andincubated with 1/1000 dilution of secondary antibody in BSA 3% (goatanti-mouse IgG Alexa Fluor 488 and goat anti-rabbit Alexa Fluor 594,Invitrogen) for 45 min at room temperature. Images are taken with aconfocal microscope at 60× magnification.

Example 2 Potential Morphology Specific Reagents to TDP-43 that couldDistinguish Between Amyotrophic Lateral Sclerosis and FrontotemporalDementia

In order to isolate therapeutic agents against brain-derived TDP-43(found in FTD and ALS patients), a combination of phage displaytechnology and AFM was used. A mixture of Tomlinson I and J and Sheetsphage libraries were used for the initial screening (10¹² pfu/mL). Aseries of negative panning steps were executed to eliminate phageparticles that were reactive with bovine serum albumin (BSA),α-synuclein (α-syn), healthy tissue and TDP-43 isolated from healthypatients. 20 μm and 5 μm AFM images were taken before and after thepanning process. (FIGS. 4A-4D)

Twelve rounds of negative panning against 1 mg/mL of Bovine SerumAlbumin (BSA) were carried out using immunotubes. Once BSA specificbinders was excluded, eight rounds of negative panning were performedagainst 500 μg/mL and 100 μg/mL of α-syn in immunotubes. Subsequently,binders to healthy tissue were removed using 50 μg/mL and 10 μg/mL ofhomogenized healthy human brain tissue samples. Lastly, eight rounds ofnegative panning were carried out against 5 μg/mL of TDP-43immunoprecipitated from healthy human brain tissue.

After the negative panning steps, simultaneously, but separately, tworounds of positive panning were completed with 10 μg/mL of TDP-43isolated from the brains of ALS patients and two rounds using FTDisolated TDP-43. The unbound phage after the second round of panningagainst ALS TDP-43 was collected and used for positive panning againstFTD TDP-43 and vice versa. This allowed for the isolation of FTD and ALSTDP-43 specific clones.

Example 3 Characterization of ALS Clones

Forty-five clones that may react specifically to the TDP-43 proteinimmunoprecipitated from the brains of ALS patients were isolated usingthe AFM biopanning process. DNA sequencing results reduced the number ofpotential clones to 40. In the initial characterization, the TDP-43protein immunoprecipitated from ALS, FTD and healthy individuals, aswell as healthy brain tissue homogenate were immobilized on mica. Thesetargets were then incubated with phage particles produced using eachindividual clone and imaged using AFM. The AFM generated images furthersuggested which clones may be ALS specific. Some of the potential clonesfrom the screening process are presented in FIGS. 5A-5C. Significantphage reactivity to TDP-43 from ALS patients is expected while little tono binding should be detected with the TDP-43 from FTD and healthyindividuals and the healthy brain tissue homogenate. Both 5 μm and 20 μmimages are illustrated in some cases to clearly exhibit phage binding.Further characterization of the clones using techniques such as ELISA,Western blotting, etc., is necessary to verify ALS TDP-43 reactiveclones.

Exemplary DNA Sequences of ALS TDP-43 reactive clones are providedbelow:

Sequence 1 (Clone 1B): (SEQ ID NO: 1)5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCCGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTTCTGCTTCTGGTACTTATACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTTCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATAATTATGCTCCTTATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′Sequence 2 (Clone 3B): (SEQ ID NO: 2)5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTCGCCATGCTGGTCAGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATATGGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCAGCGTACGAAGCCTCCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ Sequence 3 (Clone 3H):(SEQ ID NO: 3) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTGCTTCTGCTGGTACTGATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATACTACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCAAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTACTTATGCTCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′Sequence 4 (Clone 4F): (SEQ ID NO: 4)5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTATTCTAGTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGC-3′Sequence 5 (Clone 5E): (SEQ ID NO: 5)5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAATAATGCTGGTATGATACAAATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATAATGCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATTCTGCTCCTGGTACGTTCGGCCAAGGGACCAAGGTGGA AATCAAACGGGCGGCCGC-3′Sequence 6 (Clone 8D): (SEQ ID NO: 6)5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATAATAGTGGTACTTCTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTAATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGCTGCTGATCCTACTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′

Example 4 Characterization of Potential FTD Clones

About 100 clones were obtained after the three rounds of panning usingthe FTD TDP-43. Sequence verified clones were further screened using AFMagainst 5 μg/mL each of FTD TDP-43, ALS TDP-43 and healthy tissue. Weexpect to see little to no binding of these clones to the healthytissue, healthy protein and ALS TDP-43 against which negative panningwas performed. Potential clones are also expected to show a great dealof binding to the FTD TDP-43 target. AFM images of a representativeclone are provided in FIG. 6.

Example 5 Single Chain Variable Fragments Isolated Utilizing ALS TDP-43

Single chain variable fragments (scFvs) against ALS immunoprecipitated(IP) TDP-43 were previously isolated using a novel AFM based biopanningtechnology (Example 3 above). Protein translation of the DNA sequencesof the 40 ALS TDP-43 clones revealed 23 clones with no stop codons ormutations, which were then used in further studies.

1B (SEQ ID NO: 1) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCCGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTTCTGCTTCTGGTACTTATACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTTCTTCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATAATTATGCTCCTTATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′1D (SEQ ID NO: 7) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGGTATTTCTAGTTCTGGTAATACTACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGGTGCTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATGGTTCTCCTGATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′1E (SEQ ID NO: 8) 5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGGTGATACTAATCCTAGTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGC-3′ 1F(SEQ ID NO: 9) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTTCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGCTAGTTCTGGTGATGATACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTGCTAGTAGTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTAATGGTAATCCTAATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′1G (SEQ ID NO: 10) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTTCTAATGATGGTAATAATACAGATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTGATTCTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGCTGATAGTCCTACTACGTTTGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′2A (SEQ ID NO: 11) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGATATTGGTGGTGATGGTTATAATACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAACTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTTATACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGATACTAATGGTCCTTCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′2C (SEQ ID NO: 12) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTTATTCTGATGGTGGTGCTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCTACTGCTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTTCTACTAGTCCTAGTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′2D (SEQ ID NO: 13) 5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTGCATCCTCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGCTAATGCTCCTAATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGC-3′ 2F(SEQ ID NO: 14) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAATATTGCTGGTAATGGTTCTTATACATATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATGATGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTGCTACTACTCCTAATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′3A (SEQ ID NO: 15) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTAGAGTGGGTCTCAACTATTAATACTGCTGGTAATGGTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGGTACTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGGTCTATTCTGCATCCGCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCTGGTGATAGTCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′3B (SEQ ID NO: 2) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTCGCCATGCTGGTCAGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATATGGCATCCCGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGCAGCGTACGAAGCCTCCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGCCGC-3′ 3C (SEQ ID NO: 16)5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATTATAATTCTCCTTATACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGC-3′ 3D(SEQ ID NO: 17) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGATATTGATGGTGCTGGTAGTGATACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTACTAGTACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTACTACTAATCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′3H (SEQ ID NO: 3) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTGCTTCTGCTGGTACTGATACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATACTACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCAAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTACTTATGCTCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′4A (SEQ ID NO: 18) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATATATTAATGGTGATGGTGATAGTACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTAATACTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTCTTCTGCTACTCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′4B (SEQ ID NO: 19) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTGGTGCTTCTGGTAATGCTACAGCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTACTACTGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCATCCGGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCAGCTAATTATCCTACTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′4F (SEQ ID NO: 4) 5′-CCATGGCCGAGGTGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTATTCTAGTCCTTCTACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGG CGGCCGC-3′ 4G(SEQ ID NO: 20) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGCTATTGCTTCTGATGGTACTAATACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTGATGGTGATTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGCCATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTGCATCCTATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGACTGCTGCTAGTCCTAATACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′5C (SEQ ID NO: 21) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTGCTGCTGGTGGTGATAATACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGATTCTTATACTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCACCTAAGCTCCTGATCTATTCTGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTACTACTACTCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′5E (SEQ ID NO: 5) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTAATAATGCTGGTATGATACAAATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAATAATGCTTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGGTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATTCTGCTCCTGGTACGTTCGGCCAAGGGACCAAGGTGGA AATCAAACGGGCGGCCGC-3′5H (SEQ ID NO: 22) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGACTGGAGTGGGTCTCATCTATTTCTTCTACTGGTTCTGATACAACTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCTGGTTCTTCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAATTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTCTTATTCTCCTTCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′8D (SEQ ID NO: 6) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAACTATTAATAATAGTGGTACTTCTACAAATTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTACTAATTATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCAGTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAATGCTGCTGATCCTACTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′8E (SEQ ID NO: 23) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGNCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTGCTAGTTCTGGTACTAGTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGCTACTAATTCTTTTGACTATTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATAATGCATCCACTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCANCNNNAAGATTTTGCAACTTACTACTGTCAACAGACTGATGCTTCTCCTTGTACGTTCGGCCAAGGGANCAAGGTGG AAATCAAACGGGCGGCCGC-3′

In the present characterization process, phage particles and scFvs wereproduced from each clone and tested in indirect ELISAs for specificityto ALS. Brain tissue from ALS, FTD and healthy human patients wereimmobilized on high binding ELISA plates and probed with either thephage particle or scFv. Results indicated that 18 of 23 clones displayeda preference for ALS versus FTD or healthy tissue utilizing the phageparticles (FIG. 7), and four of 23 clones displayed a preference for FTDversus ALS. The data is presented as a ratio of ALS to healthy tissueand ALS to FTD. The following clones displayed a preference for ALS: 8E,1B, 2C, 3H, 2F, 5H, 2D, 1F, 3D, 5C, 1E, 1G, 3A, 4F, 4A, 4B, 3B, and 5E.

FIG. 8 provides the results observed with scFvs. Application of anotherimmunological assay, dot blots, additionally supported a preference forALS samples as demonstrated using clone 2A (FIG. 9). Both the ELISA anddot blot results indicated the preference of clones for ALS human braintissue.

Production of phage particles for the different clones was visualizedusing AFM. To demonstrate scFv production, dot blot analysis wasconducted of the un-concentrated supernatant. Some of the dot blotsrevealing scFv secretion into the supernatant are exhibited in FIG. 10.This is important since the basis of the phage capture ELISA system isthe immobilization of these scFvs on the well to capture the targets ofinterest. The bound targets were then visualized using a monomer scFv(binds all forms of the target) attached to a phage particle. In anadditional series of biopanning experiments, multiple potential monomerphages were isolated. The phage library that was negatively pannedagainst bovine serum albumin and aggregated alpha-synuclein waspositively panned against healthy IP TDP-43, ALS IP TDP-43 and FTD IPTDP-43 proteins. After each positive panning step, the bound phages wereglycine-eluted and utilized in the next round of positive panning. Thisensured that the remaining phages after all the positive panning stepswere reactive with multiple forms of TDP-43. ELISA analysisdemonstrating reactivity of these monomer phages (clones #2, #5, #3, #39and #41) with all forms of TDP, i.e., TDP from ALS, FTD and healthyhuman brain tissue (FIG. 11). The following are the DNA sequences of theTDP-43 monomer clones:

#2 (SEQ ID NO: 24) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAATATTTATAATGATGGTACTAGTACAGATTACGCAGACTCCGTGAAGGGCAGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATCTTCTAGTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCANCGGCGGNGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATACTGCATCCTCTTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGTATGATAGTACTCCTACTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′#5 (SEQ ID NO: 25) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACGGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAGATATTGCTAATACTGGTGATGCTACAGGTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAACTGGTGCTGCTTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATGCTGCATCCACTTTGCAAAGTGGGGTCCCGTCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGAGTTCTAGTGATCCTGCTACGTTCGGCCAAGGGACCAAGGTGG AAATCAAACGGGCGGCCGC-3′#3 (SEQ ID NO: 26) 5′-CCATGGCCCAGGTGTAGCTGGTGCAGTCTGGGGGAGGCTCGGTCCAGCCGGGGGGGTCCCTGAGACTCTCCTGTGTAGACTCTGGATTCACCTTCAATAGCTCTGGCATGCACTGGGTCCGCCAGGCTCCAGGCAAGGGGCTGGAGTGGGTGGCGTTTATACGCCATGATGGAAGTAAGAAATACTATGCAGACTCCGTGAAGGGCCGATTCACCATCTCCAGAGACAGTGCCAAGAACACTCTGTATCTGCAAATGGACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAGACATGGGAGCGACTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCCTCAGGTGGAGGCGGTTCAGGCGGAGGTGGCTCTGGCGGTGGCGGATCGTCTGAGCTGACTCAGGACCCTGCTGTGTCTGTGGCCTTGGGACAGACAGTCAGAATCACATGCCAAGGAGACAGCCTCAAAAGCTACTATGCAAGTTGGTACCAGCAGAAGCCAGGACAGGCCCCTGCACTTGTCATCTATGGTAAAAACAACCGGCCCTCAGGGATCCCAGACCGATTCTCTGGCTCCAGCTCAGGAAACACAGCTTCCTTGACCATCACTGGGACTCTGGCGGAAGATGAGGCTGACTATTACTGTAACTCCCGGGACAGCAGTGGTAACCGTGTGCTATTCGGCGGAGGGACCNAGCTGACCG TCCTANGTGCGGCCGC-3′ #39(SEQ ID NO: 27) 5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCAAGTATTACTTCTTCTGGTGATTCTACATCTTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAATATAGTAGTGATTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCGCTTTGCAAAGTGGGGTCCCAACAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCAACAGGCTAATACTACTCCTACTACGTTCNGNCCAAGGGNCCNAGGTGGAAATCAAACGGGCNGGCCGCACATCATCATCACCATCACGGGGCNNCAGAANAAAAACTCATCTCANAANANNNTCTGAATGGGGCCGNATAGACTGNTGAAAGTNGTTTAGCAAANNTNNTACNGAAAATTCATTTACTANGTCTGGAAAGACGANNAANTTTNNATCGTNACNCTNNCTNNNNNNNTGNCNGNNNNNGCTACNNNNNNNNNNNNNACNGNGA-3′ #41 (SEQ ID NO: 28)5′-CCATGGCCGAGGTGCAGCTGTTGGAGTCTGGGGGAGGCTTGGTACAGCCTGGGGGGTCCCTGAGACTCTCCTGTGCAGCCTCTGGATTCACCTTTAGCAGCTATGCCATGAGCTGGGTCCGCCAGGCTCCAGGGAAGGGGCTGGAGTGGGTCTCATCTATTTAGCCTACTGGTAGGCGGACATCGTACGCAGACTCCGTGAAGGGCCGGTTCACCATCTCCAGAGACAATTCCAAGAACACGCTGTATCTGCAAATGAACAGCCTGAGAGCCGAGGACACGGCCGTATATTACTGTGCGAAAAGTCGTACGCCGTTTGACTACTGGGGCCAGGGAACCCTGGTCACCGTCTCGAGCGGTGGAGGCGGTTCAGGCGGAGGTGGCAGCGGCGGTGGCGGGTCGACGGACATCCAGATGACCCAGTCTCCATCCTCCCTGTCTGCATCTGTAGGAGACAGAGTCACCATCACTTGCCGGGCAAGTCAGAGCATTAGCAGCTATTTAAATTGGTATCAGCAGAAACCAGGGAAAGCCCCTAAGCTCCTGATCTATTCTGCATCCATCTTGCAAAGTGGGGTCCCATCAAGGTTCAGTGGCAGTGGATCTGGGACAGATTTCACTCTCACCATCAGCAGTCTGCAACCTGAAGATTTTGCAACTTACTACTGTCNACAGANTACGCCTGCTCCTGGGACGTTCGGCCAAGGGACCAAGGTGGAAATCAAACGGGCGGNCGCACATCATCATCACCATCACGGGNNNGCAGAANAAAAACTCATCTCNNNNNNNATCTGAATGGGGCNNCNTANACTGNTGAAAGTGTTAGCNAANNTNNNNNNGAAAANTNNNTTNCTAACNNCTN GAANA-3′

For three out of the five clones (#5, #3 and #39), the binding patternacross the three different brain tissue samples is similar to theresults utilizing the commercial TDP-43 antibody. Application of theentire phage capture ELISA process using scFvs from six of the clonesand the potential monomer phages with ALS, FTD and healthy human braintissue produced greater signal ratios with ALS versus FTD for bothmonomer phages #3 and #5 (FIGS. 12A and B). The monomer phage was taggedwith a signal moiety, and then utilized to determine the presence of anyof the captured variants.

To ascertain whether the target bound to the capture scFvs is a TDP-43protein, a commercial TDP-43 antibody was utilized instead of themonomer phage for detection. Elevated signals were detected for mostclones with ALS tissue compared to FTD (FIG. 12C). Carrying out similarexperiments as in FIGS. 12A, B and C but with TDP-43 proteins that wereimmunoprecipitated (IP) from ALS, FTD and healthy human brain tissueagain showed a preference for ALS IP TDP-43 compared to FTD IP TDP-43for both monomer phages #3 and #5 (FIGS. 13A and B) and the commercialTDP-43 antibody (FIG. 13C). The signals with monomer phage #5 wereconsiderably greater than phage #3 and therefore monomer phage #5 seemedlike a better choice.

In previous ELISAs the inventors used either pooled or one brain samplefrom each of the three groups. To determine which clones would displayreactivity with multiple samples from each group six clones were testedwith four ALS, FTD and healthy human brain tissue individually. All sixclones exhibited greater reactivity with all four ALS samples comparedto FTD in this phage capture ELISA (FIG. 14). The pattern and level ofreactivity varied across the clones suggesting that clones were reactivewith different conformations of TDP-43. In a preliminary competitionphage capture ELISA experiment, the wells were coated with the differentscFvs and then incubated with ALS, FTD or healthy human brain tissue asusual. However, prior to this step each of the different brain tissuewere incubated in solution with the different scFvs. The scFvs recognizeand bind their targets. When the brain tissue is then added to the ELISAwells with the already bound capture scFv if the capture scFv recognizesthe same target as the scFv with which the tissue was previouslyincubated with, there is no signal during detection since binding wasprevented. The results of three of the clones are shown in FIGS. 15A, Band C. The bar labeled no competition means that the tissue added tothis well was not incubated with any scFv and therefore represent thebaseline binding for that particular scFv. The binding of these threescFvs were not inhibited by their four competitive clones, suggestingreactivity with different TDP-43 epitopes.

Since these 23 clones were initially isolated for binding to ALS TDP-43an indirect phage ELISA was carried out using pooled ALS plasma samples.The binding to ALS plasma relative to the controls for some of theclones are illustrated in FIGS. 16 A, B and C. ALS C9ORF72 is a geneticmutation thought to be related to familial ALS. Overall, elevatedbinding to ALS compared to ALS COORF72 was observed with the threeclones.

Example 6 Single Chain Variable Fragments Isolated Utilizing FTD TDP-43

Clones were isolated that preferentially bound TDP-43 present in FTDcompared to ALS brain tissue. Out of the 100 clones isolated after thepositive panning against FTD IP TDP-43, approximately 70% of the cloneshad a complete sequence without any stop codons or frameshift mutations.

Clones that were able to differentiate FTD over ALS and healthy tissuewere further transformed into competent HB cells to generate scFvs.Sequence was verified and supernatant (NOT concentrated) produced totest these clones in a sandwich ELISA. Most of the clones showed highbinding to FTD over healthy tissue and ALS confirming that the negativepanning worked (FIG. 17).

The scFv was also purified using a protein A column in a FPLC machine.SDS-PAGE gel and Western blot analyses verified that the scFv producedwas of the correct molecular weight of ˜29 kDa (FIG. 18).

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All publications, patents and patent applications are incorporatedherein by reference. While in the foregoing specification this inventionhas been described in relation to certain embodiments thereof, and manydetails have been set forth for purposes of illustration, it will beapparent to those skilled in the art that the invention is susceptibleto additional embodiments and that certain of the details describedherein may be varied considerably without departing from the basicprinciples of the invention.

The use of the terms “a” and “an” and “the” and similar referents in thecontext of describing the invention are to be construed to cover boththe singular and the plural, unless otherwise indicated herein orclearly contradicted by context. The terms “comprising,” “having,”“including,” and “containing” are to be construed as open-ended terms(i.e., meaning “including, but not limited to”) unless otherwise noted.Recitation of ranges of values herein are merely intended to serve as ashorthand method of referring individually to each separate valuefalling within the range, unless otherwise indicated herein, and eachseparate value is incorporated into the specification as if it wereindividually recited herein. All methods described herein can beperformed in any suitable order unless otherwise indicated herein orotherwise clearly contradicted by context. The use of any and allexamples, or exemplary language (e.g., “such as”) provided herein, isintended merely to better illuminate the invention and does not pose alimitation on the scope of the invention unless otherwise claimed. Nolanguage in the specification should be construed as indicating anynon-claimed element as essential to the practice of the invention.

Embodiments of this invention are described herein, including the bestmode known to the inventors for carrying out the invention. Variationsof those embodiments may become apparent to those of ordinary skill inthe art upon reading the foregoing description. The inventors expectskilled artisans to employ such variations as appropriate, and theinventors intend for the invention to be practiced otherwise than asspecifically described herein. Accordingly, this invention includes allmodifications and equivalents of the subject matter recited in theclaims appended hereto as permitted by applicable law. Moreover, anycombination of the above-described elements in all possible variationsthereof is encompassed by the invention unless otherwise indicatedherein or otherwise clearly contradicted by context.

1. An antibody or antibody fragment that specifically recognizes TDP-43associated with frontotemporal dementia (FTD), but not TDP-43 associatedwith amyotrophic lateral sclerosis (ALS) or TDP-43 associated withhealthy human brain tissue.
 2. An antibody or antibody fragment thatspecifically recognizes TDP-43 associated with amyotrophic lateralsclerosis (ALS), but not TDP-43 associated with frontotemporal dementia(FTD) or TDP-43 associated with healthy human brain tissue.
 3. Theantibody or antibody fragment of claim 1 or 2, wherein the antibody orantibody fragment is isolated according to a method comprising the stepsof: (a) a negative panning of a scFV phage library wherein the negativepanning eliminates phage that bind to non-desired antigens wherein thenegative panning comprises serially contacting phage with: (i) a genericprotein and/or α-synuclein (α-syn); and (ii) TDP-43 from healthy humanbrain tissue; and monitoring the binding of the phage to the genericprotein and the TDP-43 from healthy human brain tissue using AtomicForce Microscope (AFM) Imaging and repeating steps (i) and (ii) until nophage is observed binding to antigen by the AFM imaging to produce analiquot of phage; (b) contacting the aliquot of phage with TDP-43associated with ALS or FTD and incubating for time sufficient to allowbinding of phage to the oligomers; and (c) eluting the bound phageparticles from step (b).
 4. The antibody or antibody fragment of claim3, wherein the step of observing of the binding of the phage to theantigen is by using Atomic Force Microscope (AFM) Imaging.
 5. (canceled)6. The antibody or antibody fragment of any one of claims 3-5, whereinthe TDP-43 associated with ALS or FTD is TDP-43 associated with ALS. 7.The antibody or antibody fragment of any one of claims 3-5, wherein theTDP-43 associated with ALS or FTD is TDP-43 associated with FTD.
 8. Theantibody or antibody fragment of any one of claims 3-6, wherein thegeneric protein is bovine serum albumin (BSA).
 9. The antibody orantibody fragment of any one of claims 4-8, wherein the negative panningis repeated until less than 0-10% phage was observed by AFM imaging asbinding to antigen in step (a).
 10. The antibody fragment of any one ofclaims 1-9, wherein the antibody fragment does not contain the constantdomain region of an antibody.
 11. An antibody fragment comprising anamino acid sequence encoded by a nucleic acid, wherein the nucleic acidhas at least 80% identity to SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQID NO:4, SEQ ID NO:5, SEQ ID NO:6 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9,SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14,SEQ ID NO:15, SEQ ID NO:16, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19,SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, or SEQ ID NO:23.
 12. A bindingmolecule that binds to TDP-43 associated with ALS and does not bindTDP-43 from healthy human brain tissue or TDP-43 associated with FTD,wherein the binding molecule comprises an amino acid sequence encoded bya nucleic acid, wherein the nucleic acid has at least 80% identity toSEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ IDNO:6 SEQ ID NO:7, SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO:10, SEQ ID NO:11,SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO:14, SEQ ID NO:15, SEQ ID NO:16,SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21,SEQ ID NO:22, or SEQ ID NO:23.
 13. A method of binding TDP-43 associatedwith ALS comprising contacting a composition that comprises TDP-43associated with ALS with an antibody, antibody fragment or bindingmolecule of any one of claims 2 to 6 or 8 to
 12. 14. The method of claim13, wherein the TDP-43 associated with ALS is in a cell.
 15. The methodof claim 13, wherein the TDP-43 associated with ALS is in brain tissue.16. A method of binding TDP-43 associated with FTD comprising contactinga composition that comprises TDP-43 associated with FTD with anantibody, antibody fragment or binding molecule of any one of claim 1,3-5, or 7-10.
 17. The method of claim 16, wherein the TDP-43 associatedwith FTD is in a cell.
 18. The method of claim 16, wherein the TDP-43associated with FTD is in brain tissue.
 19. A method of detecting thepresence of TDP-43 associated with FTD in a physiological samplecomprising contacting a sample with a composition comprising anantibody, antibody fragment or a binding molecule of any one of claim 1,3-5, or 7-10 and determining the binding of the composition with thetissue sample wherein binding of the composition to the tissue sample isindicative of the presence of TDP-43 associated with FTD in the tissuesample wherein the presence of the TDP-43 associated with FTD isindicative of frontotemporal dementia.
 20. A method of detecting thepresence of TDP-43 associated with ALS in a physiological samplecomprising contacting a sample with a composition comprising anantibody, antibody fragment or a binding molecule of any one of claim2-6 or 8-12 and determining the binding of the composition with thetissue sample wherein binding of the composition to the tissue sample isindicative of the presence of TDP-43 associated with ALS in the tissuesample wherein the presence of the TDP-43 associated with ALS isindicative of amyotrophic lateral sclerosis.
 21. The method of claim 19or 20, wherein the physiological sample is brain tissue, serum,cerebrospinal fluid (CSF), blood, urine or saliva.
 22. A method oftreating FTD or ALS in the brain of a mammal comprising administering tothe mammal a composition comprising an antibody, antibody fragment or abinding molecule of any one of claims 1 to
 12. 23. An imagingcomposition specific for TDP-43 associated with ALS or FTD comprising anantibody, antibody fragment or a binding molecule of any one of claims 1to 12 conjugated to an imaging agent.